1. Field of the Invention
This invention relates to the creation, optimization and use of new thermostable esterase enzymes. More particularly, the invention relates to thermostable enzymes optimized to remove ester-linked para-nitrobenzyl (pNB) protecting groups from carboxyl functional groups on b-lactam antibiotics and other compounds. This invention also relates to methods by which such enzymes can be altered and optimized for specific substrates and reaction conditions. Also, this invention relates to purifying thermostable enzymes based on their ability to withstand relatively high temperatures.
2. Description of Related Art
The publications and other reference materials referred to herein to describe the background of the invention and to provide additional details regarding its practice are hereby incorporated by reference. For convenience, the reference materials are numerically referenced and identified in the appended bibliography.
Efficient protection and deprotection of functional groups is critical to successful organic synthesis of polyfunctional molecules. Synthetic schemes often require that a given functional group be protected or deprotected selectively, under the mildest conditions and in the presence of functional groups of similar reactivity or other structures that are sensitive to acids, bases, oxidation and reduction. These situations represent severe problems for the synthesis of complex, polyfunctional molecules which cannot, or only with great difficulty, be solved using classical chemical tools.
The array of protecting group techniques can be substantially enriched by the application of enzymes. Enzymes can discriminate stereoisomers as well as offer the opportunity to carry out highly chemo- and regioselective transformations. The highly selective nature of enzymes may be exploited advantageously in the manipulation of protecting groups and in the synthesis of chiral compounds such as drugs and natural products. Furthermore, enzymes function under mild conditions, often operating at or near room temperature and at neutral, weakly acidic or weakly basic pH values. In many cases they combine a high selectivity for the reactions they catalyze with a broad substrate tolerance. Therefore, the application of enzymes can be viable alternatives to classical chemical protection/deprotection methods for the introduction and/or removal of suitable protecting groups (1). The introduction of new enzymes with reactivities and temperature tolerances differing from existing enzymes is highly desirable.
Carboxy groups are often protected by conversion to the benzyl or para-nitrobenzyl (pNB) esters (2). Benzyl esters are resistant to treatment with reagents such as trifluoroacetic acid, triethylamine, diisopropylethylamine, but are readily removed by hydrogenolysis (over a Pd catalyst). Hydrogenolysis is not appropriate, however, for compounds containing double bonds, azides, imines, or activated aldehydes, or other functional groups that will be reduced. Benzyl esters can also be cleaved using a zinc catalyst under anhydrous conditions, but the extent of hydrolysis is variable and dependent on the conditions (e.g., time and temperature) of the reaction. The reaction must be carried out under anhydrous conditions, in an organic solvent. Both the organic solvent and catalyst can give rise to toxicity or disposal problems for large-scale reactions.
Modification by substitution in the aromatic ring can alter the sensitivity of the benzyl group towards deprotection by acidic reagents. PNB esters display increased resistance to acid hydrolysis.
During total synthesis or chemical modification of an antibiotic, several sites on the antibiotic could be adversely affected by the reagents used to carry out any given reaction step. Para-nitrobenzyl alcohol (pNB-OH) is commonly used to protect carboxylic acid functionalities in cephalosporin-derived antibiotics (U.S. Pat. No. 3,725,359 1975!) (3). The pNB ester linkage is stable enough to withstand the various reaction conditions used in subsequent chemical steps. After chemical synthesis is completed, deprotection is required to return the cephalosporin-pNB ester to its original and active carboxylic acid form. The chemistry used to deprotect the carboxylic acid involves a catalytic form of zinc in concentrated organic solvent, and on an industrial scale this process generates large amounts of solvent and zinc-containing waste material. Cost is associated with processing of waste to make it safe for disposal. In 1975, scientists at Eli Lilly & Co. interested in pursuing alternative methods of deprotection for higher yield and lower disposal costs began a search for an esterase capable of performing this deprotection reaction (3).
The enzyme known as para-nitrobenzyl esterase (pNB esterase) was discovered in 1975 by scientists at Eli Lilly & Co., who screened whole cell preparations of numerous bacterial and fungal cultures for those possessing catalytic activity toward the hydrolysis of a p-nitrobenzyl protected cephalosporin (3). A Bacillus subtilis culture (NRRL B8079) showed the highest catalytic activity toward two cephalosporin-derived pNB-protected substrates of all the cultures tested. Although the reaction yield was high, the enzyme activity was not sufficient to consider for industrial application.
A chromatographically pure solution of pNB esterase was isolated at Eli Lilly, and its amino acid sequence partially determined. Using this partial sequence, DNA primers were constructed and used to isolate and sequence the gene for pNB esterase. This gene was cloned into E. coli, where it was over-expressed to produce pNB esterase in large quantities (4). However, partially purified enzyme preparations of "pNB esterase" could not compete with the speed, economy, or the small reaction volumes (due to lack of solubility of substrate in the purely aqueous environments preferred by the enzyme) of the zinc-catalyzed deprotection reaction.
The targeted reaction substrates have changed over the fifteen year period as well. Cephalosporin-derived antibiotics continued to evolve from the first generation cephalexin (one of the two original cephalosporin substrates used to search for pNB esterase), second generation cefaclor, third generation cefixime, and fourth generation loracarbef. These antibiotics have been developed to be readily absorbed (generation one), more potent (generation two), much more potent (generation three), and, finally, immensely more stable in the body (generation four) (5). They all are synthesized using the pNB ester protecting group (6). In protected form, with perhaps the exception of cefixime, all are only sparingly soluble in water.
The pNB esterase enzyme has been further characterized (6). It is a water soluble, monomeric serine esterase of 54 kD molecular weight and a pI of 4.1. The enzyme is active on a variety of ester substrates, ranging from the cephalosporin-derived compounds on which it was screened to a number of simple organic esters. Reported K.sub.M values for cephalosporin-derived substrates are in 0.5 to 2 mM range. The enzyme functions best at temperatures below 50.degree. C., and its pH optimum is between 8 and 9.
The pNB esterase still suffers from a problem common to a large number of enzyme reactions in the performance of synthetic chemistry: the desired substrates are only sparingly soluble in water, and the enzyme's catalytic ability is drastically reduced by even small quantities of water-miscible non-aqueous solvents.
It was discovered that substitution of amino acids at one or more specific amino acid positions resulted in the formation of enzymes having improved capabilities in aqueous and aqueous-organic media (43, 44). The specific amino acid position numbers at which substitutions were made to achieve these modified para-nitrobenzyl esterases were position Nos. 60, 94, 96, 144, 267, 271, 322, 334, 343, 358 and 370.
Specific amino acid substitutions have been disclosed which provide specific modified para-nitrobenzyl esterases having improved stability and/or ester hydrolysis activity in organic media (43, 44). The specific amino acid substitutions include Ile 60 Val, Ser 94 Gly, Asn 96 Ser, Leu 144 Met, Lys 267 Arg, Phe 271 Leu, His 322 Arg, Leu 334 Val, Leu 334 Ser, Ala 343 Val, Met 358 Val, and Tyr 370 Phe. One or more of these specific substitutions were found to increase the enzymatic activity and/or stability of the esterases in aqueous and aqueous-organic media. Ten specific modified para-nitrobenzyl esterases were disclosed which show enhanced activity in aqueous or aqueous-organic media over naturally occurring para-nitrobenzyl esterase. The amino acid sequences for these modified esterases are set forth in SEQ. ID. NOS. 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22. These variants were identified as variants 1-1h9, 2-19e10, 3-10c4, 4-38b9, 4-43e7, 4-54b9, 2-13f3, 2-23e1, 4-53d5 and 5-1a12, respectively. The naturally occurring esterase is identified as O-Wtpnb or WT and is set forth in SEQ. ID. NO. 2.
Natural enzymes, such as pNB esterase, are poised on the brink of conformational instability, with native structures that walk a tightrope between large stabilizing and destabilizing forces. The molecular origins of enzyme stability are critical to understanding how proteins fold into their unique three-dimensional structures as well as to understanding the limits of (protein-based) life. Life on earth exists over a wide temperature range--nearly 200.degree. C.--yet proteins isolated from organisms inhabiting the very coldest and hottest environments do not differ from one another in anything but the most subtle ways (28).
Enhancing the stability of enzymes is key to improving them for a wide range of applications, including catalysts in chemical processes and additives for laundry detergents. It has long been hoped that studies of naturally thermostable proteins would yield general rules that could be applied to stabilizing other, less-stable proteins. The extreme thermostability of many enzymes with significant half lives at high temperatures--even 100.degree. C. and above--is often defined within their amino acid sequences rather than by extrinsic factors. Therefore the amino acid sequences and structures of enzymes from mesophilic organisms (i.e. optimal growth temperature (T.sub.opt approximately 20-50.degree. C.) have been compared to those from thermophiles (T.sub.opt approximately 50-80.degree. C.) and extreme thermophiles (T.sub.opt greater than or equal to 80.degree. C.) in an effort to identify the interactions responsible for conferring enhanced thermostability (29; 30; 31). Numerous and intensive site-directed mutagenesis efforts have also probed this issue (32 and 33). Despite these efforts, considerable disagreement remains over which forces dominate thermostabilization mechanisms, and no generally-applicable rules for thermostabilizing proteins have been established. Some of the confusion arises from the large evolutionary distances that separate thermophilic enzymes from their mesophilic homologs. The relatively few mutations responsible for differences in thermostability are not easily identified in a background of many (often 100 or more) neutral mutations. Moreover, substantial increases in thermostability are often the result of multiple mutations, each of which makes a small but cumulative contribution. Another, more fundamental, reason is that the effects of temperature on the forces contributing to protein stability are many and highly complex (34). Any rules for engineering protein stability are likely to be protein-specific, and such efforts will need to be guided by detailed 3-dimensional structural information (35).
The design problem becomes even more challenging if improvements in thermostability are not to come at the cost of decreases in enzyme activity, particularly at reduced temperatures. It is widely believed that enhanced molecular rigidity is a prerequisite for thermostability, while maintaining flexibility is required for catalytic activity. The fact that natural thermophilic enzymes are active and stable at higher temperatures, but their activities at lower temperature are often compromised, has been used to support the idea that an improvement in one property (stability) will come at the cost of the other (activity) (33). An alternative explanation for the observation that natural proteins from thermophiles are less active than their mesophilic counterparts at the lower temperatures, however, is that natural selection has exerted pressure on one, but not both these properties. Because enzymes from a thermophile need not be active at low temperature, this property is free to drift. Thus the low activities of thermophilic enzymes at mesophilic temperatures may not necessarily mean that high activity is incompatible with high thermal stability.
In view of the above situation, there is a continuing need to develop new enzymes which have expanded catalytic capabilities. In particular, new thermostable enzymes are needed which can be used to provide ester cleavage for a variety of substrates and settings, including polar non-aqueous solvents.